Yoichi Miyamoto

Showa University, Shinagawa, Tōkyō, Japan

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Publications (55)170.26 Total impact

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    ABSTRACT: Background Rheumatoid arthritis (RA) is an inflammatory disease that leads to destruction of both articular cartilage and bone tissues. In rheumatic joints, synoviocytes and T-lymphocytes as well as bone cells produce the receptor activator of nuclear factor ¿-B (RANK) ligand (RANKL), which binds to RANK on the surface of osteoclasts and their precursor cells to induce differentiation and activation of osteoclasts. Hence, inhibition of RANKL may be a promising approach to suppress osteolysis in RA. On the other hand, RANKL production by lymphocytes indicates the possibility that its inhibition would be effective to suppress inflammation in RA. In addition, it has been reported that cathepsin K, a predominant cysteine protease in osteoclasts, is involved in cartilage destruction in RA model mice. Here, we evaluated the effects of an anti-RANKL antibody on inflammation in footpads and degradation of articular cartilage in RA model mice.ResultsWe induced arthritis in mice by injection of anti-type II collagen antibodies and lipopolysaccharide (LPS). Inhibition of RANKL by an anti-RANKL antibody (OYC1, Oriental Yeast, Tokyo, Japan) was confirmed by increased bone volume in the metaphysis of tibias. Swelling in either limb until day 14 was seen in 5 of 6 mice injected with anti-collagen antibodies and LPS without treatment with OYC1, while that was seen in 4 of 5 mice treated with OYC1. The average arthritis scores on day 14 in those groups were 2.17 and 3.00, respectively, indicating that OYC1 did not ameliorate inflammation in the limbs. Histological analyses indicated that OYC1 does not protect articular cartilage from destruction in mice with arthritis.Conclusions Our present study failed to show the effectiveness of an anti-RANKL antibody to ameliorate inflammation in the limbs or protect articular cartilage from degradation in a collagen antibody-induced arthritis mouse model.
    Journal of Negative Results in BioMedicine 12/2014; 13(1):18. DOI:10.1186/s12952-014-0018-0 · 1.47 Impact Factor
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    ABSTRACT: The functional requirements of regenerated calcified tissues are that they enable the tissues to bear a variety of imposed stress and consequent contact-induced strain without substantial fracture. Here we demonstrate the effects of glucocorticoid hormones such as dexamethasone and hydrocortisone on the nanomechanical properties of calcified nodules formed by mouse osteoblastic MC3T3-E1 cells in differentiation inducing medium containing ascorbic acid and β-glycerophosphate. Neither cell proliferation nor calcium deposition, evaluated using alizarin red and von Kossa staining was affected by dexamethasone. On the other hand, calcified nodules formed in the presence of dexamethasone were significantly harder and stiffer than those formed in their absence. In particular, a series of nanoindentation tests revealed that the calcified nodules formed in the presence of dexamethasone showed enhanced stiffness against dynamic strain as compared to a quasi-static load. Furthermore, Raman spectroscopy revealed that dexamethasone and hydrocortisone increased the apatite/matrix ratio and lowered that of carbonate in the nodules. Our results suggest that glucocorticoids are required for in vitro formation by osteoblasts of more mature calcified nodules containing apatite-phosphate.
    Acta Biomaterialia 11/2014; DOI:10.1016/j.actbio.2014.10.038 · 5.68 Impact Factor
  • Yoichi Miyamoto
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    ABSTRACT: Background Periodontitis is a chronic inflammatory disease accompanied by alveolar bone loss. Porphyromonas gingivalis, which plays a key role in the etiology of periodontitis, produces cysteine proteases called gingipains to promote proteolysis. Gingipains are classified into two groups based on their cleavage site specificity, specifically arginine-specific gingipains (Rgps) and lysine-specific gingipains (Kgps). Highlight We found that osteoclast differentiation induced by active vitamin D3, Toll-like receptor ligands including lipopolysaccharide, and inflammatory cytokines such as tumor necrosis factor-α and interleukin-1β was enhanced by a secreted Kgp in co-cultures of mouse osteoblasts and bone marrow cells, whereas RgpB had no effect on osteoclast differentiation under the same experimental conditions. The effect of Kgp on osteoclast differentiation was completely blocked by an inhibitor of Kgp. Further, osteoprotegerin (OPG), a protein that regulates osteoclast differentiation, was degraded by Kgp. Kgp-mediated osteoclast differentiation was not observed in co-cultures of OPG-deficient osteoblasts and bone marrow cells. Conclusion Our data suggests that degradation of OPG by Kgp is a crucial event in the progression of osteolysis in periodontitis.
    Journal of Oral Biosciences 07/2014; DOI:10.1016/j.job.2014.06.002
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    ABSTRACT: Objectives: It is well known that decreased insulin signaling in bone causes osteoporosis in type I diabetes patients. Insulin depletion increases blood concentrations of ketone bodies as well as that of glucose in the patients. We are interested in the role of ketone bodies in bone metabolism, which has not been studied well. In this study, we investigated the effects of ketone bodies on osteoblast phenotypes induced by bone morphogenetic protein-2 (BMP-2). Methods: We investigated the effects of acetoacetate and b-hydroxybutyrate, the major ketone bodies found in the blood, on alkaline phosphatase (ALP) activity, mineralization, and expression of differentiation marker genes in mouse osteoblastic MC3T3-E1 cells and mouse primary osteoblasts induced by BMP-2. ALP activity was assessed in both types of cells after stimulation by BMP-2 in the presence and absence of the ketone bodies. Effects of these ketone bodies on mineralization induced by BMP-2 in the presence of ascorbic acid, b-glycerophosphate, and dexamethasone were examined by alizarin red staining. Expression of osteoblast marker genes was assessed by RT-PCR and real-time RT-PCR. Results: Acetoacetate potentiated the ALP activity in a dose- and time-dependent manner from their physiological to pathological concentrations. Contrary, b-hydroxyacetate lowered ALP activity. Mineralization was also augmented by acetoacetate and inhibited by b-hydroxybutyrate, respectively. The expression for ALP mRNA induced by BMP-2 was not affected by acetoacetate or b-hydroxybutyrate, indicating that they bi-directionally control the enzyme activity of ALP in osteoblasts and hence their mineralization. On the other hand, either the ketone body did not alter the expression of marker genes of osteoblast differentiation. Conclusion: Increase in the blood concentrations of ketone bodies would modulate functions of osteoblasts but not their differentiation. The results obtained here suggest that they are important endogenous factors that regulate bone metabolism both in physiological and pathological situations.
    IADR General Session and Exhibition 2014; 06/2014
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    ABSTRACT: Periodontitis is a chronic inflammatory disease accompanied by alveolar bone resorption by osteoclasts. Porphyromonas gingivalis, an etiological agent for periodontitis, produces cysteine proteases called gingipains, which are classified based on their cleavage site specificity, i.e., arginine (Rgps) and lysine (Kgps) gingipains. We previously reported that Kgp degraded osteoprotegerin (OPG), an osteoclastogenesis inhibitory factor secreted by osteoblasts, and enhanced osteoclastogenesis induced by various Toll-like receptor (TLR) ligands (Yasuhara R, et al. Biochem J, 419, 159-166, 2009). Osteoclastogenesis is induced not only by TLR ligands but also by proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-17A, in inflammatory conditions such as periodontitis. Although Kgp augmented osteoclastogenesis induced by TNF-α and IL-1β in co-cultures of mouse osteoblasts and bone marrow cells, it suppressed that induced by IL-17A. In a comparison of proteolytic degradation of these cytokines by Kgp in a cell-free system with that of OPG, TNF-α and IL-1β were less susceptible, while IL-17A and OPG were equally susceptible to degradation by Kgp. These results indicate that the enhancing effect of Kgp on cytokine-induced osteoclastogenesis is dependent on the difference in degradation efficiency between each cytokine and OPG. In addition, elucidation of the N-Terminal amino acid sequences of OPG fragments revealed that Kgp primarily cleaved OPG in its death domain homologous region, which might prevent dimer formation of OPG required for inhibition of RANKL. Collectively, our results suggest that degradation of OPG by Kgp is a crucial event in development of osteoclastogenesis and bone loss in periodontitis.
    Journal of Biological Chemistry 04/2014; 289(22). DOI:10.1074/jbc.M113.520510 · 4.60 Impact Factor
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    ABSTRACT: Objective: Osteoclasts are bone-resorbing giant cells differentiated from a monocyte/macrophage lineage, and involved in inflammatory bone destruction accompanied by periodontitis and rheumatoid arthritis. Recent studies have shown that osteoclast precursors reside not only in bone marrow, but also peripheral blood and the spleen, though their precise characteristics in those tissues have not been elucidated. We isolated and characterized osteoclast precursors in those tissues from mice using the macrophage marker CD11b (also called Mac-1). Method: We utilized a cell separation system with magnetic bead-labeled anti-mouse CD11b antibodies to isolate CD11b+ and CD11b– cells from bone marrow, blood, and spleen specimens. Isolated cells were cultured in the presence of M-CSF (100 ng/ml) with or without RANKL (100 ng/ml), an osteoclast differentiation factor, for 5 days and their potential for osteoclastogenesis was evaluated by enzymatic staining for TRAP, an osteoclast differentiation marker. Result: In bone marrow, CD11b– (approx. 65%) but not CD11b+ (approx. 30%) cells differentiated into osteoclasts in response to RANKL stimulation. In contrast, CD11b+ (approx. 2%) but not CD11b– (approx. 93%) cells in blood differentiated into osteoclasts. In the adult (6 weeks old) spleens, neither CD11b+ (approx. 5%) nor CD11b– (approx. 95%) cells differentiated into osteoclasts, whereas both (approx. 8% and 90%, respectively) were found differentiated into osteoclasts in spleens from young mice (1 week old). Since osteoblasts support osteoclast differentiation, we cultured CD11b– cells isolated from bone marrow with the osteoblastic cell line UAMS-32 for 24 hours, after which the CD11b– cells expressed CD11b and differentiated into osteoclasts with RANKL stimulation. Conclusion: Our results suggest that cells composing bone marrow, blood, and young but not adult spleen tissues possess various types of osteoclast precursors. In addition, CD11b+ osteoclast precursors in blood may originate from CD11b– bone marrow cells after interaction with osteoblasts.
    IADR Asia/Pacific Region (APR) Regional Meeting and Co-Annual Scientific Meeting of IADR Divisions 2013; 08/2013
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    ABSTRACT: Carbonic anhydrase (CA) IX is a transmembrane isozyme of CAs that catalyzes reversible hydration of CO(2). While it is known that CA IX is distributed in human embryonic chondrocytes, its role in chondrocyte differentiation has not been reported. In the present study, we found that Car9 mRNA and CA IX were expressed in proliferating but not hypertrophic chondrocytes. Next, we examined the role of CA IX in the expression of marker genes of chondrocyte differentiation in vitro. Introduction of Car9 siRNA to mouse primary chondrocytes obtained from costal cartilage induced the mRNA expressions of Col10a1, the gene for type X collagen α-1 chain, and Epas1, the gene for hypoxia-responsible factor-2α (HIF-2α), both of which are known to be characteristically expressed in hypertrophic chondrocytes. On the other hand, forced expression of CA IX had no effect of the proliferation of chondrocytes or the transcription of Col10a1 and Epas1, while the transcription of Col2a1 and Acan were up-regulated. Although HIF-2α has been reported to be a potent activator of Col10a1 transcription, Epas1 siRNA did not suppress Car9 siRNA-induced increment in Col10a1 expression, indicating that down-regulation of CA IX induces the expression of Col10a1 in chondrocytes in a HIF-2α-independent manner. On the other hand, cellular cAMP content was lowered by Car9 siRNA. Furthermore, the expression of Col10a1 mRNA after Car9 silencing was augmented by an inhibitor of protein kinase A, and suppressed by an inhibitor for phosphodiesterase as well as a brominated analog of cAMP. While these results suggest a possible involvement of cAMP-dependent pathway, at least in part, in induction of Col10a1 expression by down-regulation of Car9, more detailed study is required to clarify the role of CA IX in regulation of Col10a1 expression in chondrocytes.
    PLoS ONE 02/2013; 8(2):e56984. DOI:10.1371/journal.pone.0056984 · 3.53 Impact Factor
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    ABSTRACT: Implantation of octacalcium phosphate (OCP), a hydroxyapatite precursor, has been reported to induce chondrogenesis in vivo. In this study, we examined the effects of OCP on the chondrogenic differentiation of mouse chondroblastic ATDC5 cells in vitro. Contrary to our expectation, chondrogenic differentiation of ATDC5 cells evaluated by the mRNA expression of Col2a1, Acan and Col10a1 was suppressed by OCP. Among Sox9, Sox5 and Sox6, essential transcription factors for chondrogenesis, the expression of Sox6 mRNA was markedly lowered by OCP. Whereas ATDC5 cells dissolved OCP to liberate calcium and inorganic phosphorus, increased calcium or phosphate in the medium had little effect on the differentiation of these cells. Direct contact of ATDC5 cells with OCP was required to suppress the expression of Col2a1 and Sox6 mRNAs, whereas the introduction of Sox6 short interfering RNA lowered the expression of Col2a1 mRNA. On the other hand, the forced expression of Sox6 protein partially but significantly, restored the expression of Col2a1 mRNA suppressed by OCP. These results indicate that OCP suppresses the chondrogenic differentiation of ATDC5 cells, at least in part, at the Sox6 transcription level.
    Cell and Tissue Research 12/2012; DOI:10.1007/s00441-012-1548-8 · 3.33 Impact Factor
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    ABSTRACT: Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK) differentiate into osteoclasts following stimulation with the RANK ligand (RANKL). Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition) or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition). BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS) and tumor necrosis factor -αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6) in BMMs induced their differentiation into osteoclasts even under the non-adherent condition. These results suggest that cell adhesion signaling regulates RANK expression in osteoclast precursors.
    PLoS ONE 11/2012; 7(11):e48795. DOI:10.1371/journal.pone.0048795 · 3.53 Impact Factor
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    ABSTRACT: Nephronectin (Npnt) is an extracellular matrix protein known to be a ligand for the integrin α8β1. We previously demonstrated that Npnt expression was suppressed by TGF-β through ERK1/2 and JNK in osteoblasts. In this study, we found that inhibition of a TGF-β type I receptor (TGF-β R1, Alk5) by a specific inhibitor {2-[3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine} strongly induced Npnt expression in osteoblast-like MC3T3-E1 cells. The Alk5 inhibitor-induced increase of Npnt expression occurred in both time- and dose-dependent manners, while that expression was also induced by introduction of an siRNA for Smad2, a central intracellular mediator of TGF-β signaling. These results suggest that the expression of Npnt is regulated by the Alk5-SMAD signaling pathway in osteoblasts.
    Biochemical and Biophysical Research Communications 07/2012; 425(2):390-2. DOI:10.1016/j.bbrc.2012.07.106 · 2.28 Impact Factor
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    ABSTRACT: Bone morphogenetic proteins (BMPs) control the expressions of many genes involved in bone formation. On the basis of our hypothesis that BMP2 stimulation-regulated gene expression plays a critical role in osteoblast differentiation, we performed genome-wide screening of messenger RNA from BMP2-treated and -untreated C2C12 cells using a DNA microarray technique. We found that the expressions of Gremlin1 and Gremlin2, which are known BMP antagonists, were bidirectionally regulated by BMP2. Gremlin1 was down-regulated by BMP2, while Gremlin2 was up-regulated in both time- and dose-dependent manners. Ablation of Gremlin1 or Gremlin2 enhanced osteoblast differentiation induced by BMP2. On the other hand, treatment with recombinant Gremlin1 inhibited BMP2-induced osteoblast differentiation. Furthermore, treatment with Smad4 siRNA and the p38 MAPK inhibitor SB203580 suppressed BMP2-induced Gremlin2 gene expression. The differential regulation of Gremlin1 and Gremlin2 gene expressions by BMP2 may explain the critical function of these genes during osteoblast differentiation.
    Calcified Tissue International 05/2012; 91(1):88-96. DOI:10.1007/s00223-012-9614-5 · 2.75 Impact Factor
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    ABSTRACT: R848, also known as resiquimod, acts as a ligand for toll-like receptor 7 (TLR7) and activates immune cells. In this study, we examined the effects of R848 on differentiation, survival, and bone-resorbing function of osteoclasts. R848 inhibited osteoclast differentiation of mouse bone marrow-derived macrophages (BMMs) and human peripheral blood-derived monocytes induced by receptor activator of NF-κB ligand in a dose-dependent manner. In addition, it inhibited mouse osteoclast differentiation induced in cocultures of bone marrow cells and osteoblasts in the presence of dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. However, R848 did not affect the survival or bone-resorbing activity of mouse mature osteoclasts. R848 also upregulated the mRNA expression levels of interleukin (IL)-6, IL-12, interferon (IFN)-γ, and inducible nitric oxide synthase in mouse BMMs expressing TLR7. IFN-β was consistently expressed in the BMMs and addition of neutralizing antibodies against IFN-β to the cultures partially recovered osteoclast differentiation inhibited by R848. These results suggest that R848 targets osteoclast precursors and inhibits their differentiation into osteoclasts via TLR7.
    Cytotechnology 02/2012; 64(3):331-9. DOI:10.1007/s10616-012-9442-5 · 1.45 Impact Factor
  • Kentaro Yoshimura, Yoichi Miyamoto, Ryutaro Kamijo
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    ABSTRACT: Periodontitis is the inflammatory disease caused by periodontal pathogens in dental plaque, and progression of this pathological condition is also considered as a major cause of alveolar bone resorption and subsequent tooth depletion. LPS and several kinds of proteinases produced by periodontal pathogens directly destroy periodontal tissues. On the other hand, host defense systems existing in the periodontal tissues exert essential roles in protection of periodontal tissues from bacterial invasion. Immune system divides broadly into 2 categories, innate immune and acquired immune systems. The former is the first defensive barrier against infectious diseases of pathogens, and phagocytic cells including macrophages and neutrophils are involved in this immune system as innate immune function. The latter is the secondary immune system that antigen-presenting cells such as dendritic cells detect smaller pathogens or intracellular pathogens, and antigens which retain as intact proteins on the surfaces of these cells activate T cells and B cells. However, once inflammation becomes persistent with bacteria, these biological defective mechanisms causes breakdown of host innate and acquired immune systems, and subsequent destruction of periodontal tissues. Here we review the mechanisms by which periodontal pathogens cause bone resorption.
    Clinical calcium 01/2012; 22(11):1685-1692.
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    ABSTRACT: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-α (TNF-α), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose-dependent manners through the nuclear factor kappa B (NF-κB) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-α in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-α-induced inhibition of osteoblast differentiation. These results suggest that TNF-α inhibits POEM expression through the NF-κB signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-α.
    Biochemical and Biophysical Research Communications 06/2011; 410(4):766-70. DOI:10.1016/j.bbrc.2011.06.048 · 2.28 Impact Factor
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    ABSTRACT: Objective: Interleukin-1β (IL-1β) acts as a key mediator of articular cartilage degeneration in rheumatoid arthritis and osteoarthritis where chondrocyte death is observed. We reported that IL-1β induced chondrocyte death through mitochondrial dysfunction and energy depletion in a reactive oxygen species (ROS)-dependent manner. We also found that IL-1β induced the expression of NOX-2, phagocyte-type NADPH-oxidase, which was required for IL-1β-induced ROS production and cell death in chondrocytes. Moreover, an increase in lactate production was observed in the same experimental settings. Here we examined the role of monocarboxylate transporters-1 (MCT-1), a lactate transporter distributed in plasma and mitochondrial inner membranes, in IL-1β-induced chondrocyte death. Methods: Mouse chondrocyte-like ATDC5 cells were incubated with IL-1β after introduction of a small interfering RNA (siRNA) for MCT-1 or its control RNA. Cell survival was evaluated by cell counting and MTT assay. Gene expressions of MCT-1 and NOX-2 were quantitatively analyzed by real-time RT-PCR. Phosphorylation and degradation of IκB and nuclear translocation of NF-κB were examined by Western blotting and immunofluorescence, respectively. Results: Introduction of MCT-1 siRNA suppressed the expression of NOX-2 mRNA as well as cell death induced by IL-1β in ATDC5 cells. BAY11-7085, an NF-κB inhibitor, reduced IL-1β-induced NOX-2 expression, indicating that NF-κB pathway plays a critical role in the NOX-2 expression induced by IL-1β in ATDC5 cells. Phosphorylation or degradation of IκB immediately after stimulation by IL-1β was not affected by MCT-1 siRNA, but degradation of IκB and nuclear translocation of NF-κB observed after prolonged exposure to IL-1β were significantly suppressed by MCT-1 siRNA. Conclusion: These results indicate that MCT-1 contributes to the cell death by induction of NOX-2 expression via late-phase activation of NF-κB in ATDC5 cells exposed to IL-1β.
    IADR General Session 2011; 03/2011
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    ABSTRACT: Objectives: POEM (preosteoblast epidermal growth factor-like repeat protein with meprin, A5 protein, and receptor protein-tyrosine phosphatase μ domain), also known as nephronectin, is an extracellular matrix protein considered to play critical roles in the development and functions of various tissues, such as those of the kidneys, bone, muscle tissues, and endocrine organs, via adhesion molecules. Recently, we found that tumor necrosis factor-α (TNF-α), which is a key regulator of bone matrix properties and composition, and inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line, MC3T3-E1. The purpose of this study was to investigate the mechanisms governing the regulation of POEM expression by TNF-α in osteoblasts. Methods: The gene expression of POEM was examined by qPCR, while osteoblast differentiation was analyzed by determining alkaline phosphatase (ALPase) activity. Results: TNF-α-induced down-regulation of POEM gene expression occurred in both time- and dose- dependent manners. TNF-α stimulates NF-κB pathway in a variety of cell lines including MC3T3-E1 cells. To determine whether NF-κB pathway is involved in the down-regulation of POEM gene expression by TNF-α, MC3T3-E1 cells were pretreated with an NF-κB activation inhibitor followed by treatment with TNF-α. Treatment of an NF-κB activation inhibitor inhibited down-regulation of POEM gene expression by TNF-α. To elucidate the relationship between inhibition of osteoblast differentiation and reduction of POEM expression by TNF-α, the expressions of ALP and osteocalcin were also examined. The expressions of those genes were down-regulated by TNF-α in a manner consistent with our findings for POEM. Conclusion: TNF-α regulates POEM gene expression via NF-κB activation in osteoblasts, as well as ALP and osteocalcin in a similar manner. Our results suggest a relationship between inhibition of osteoblast differentiation and reduction of POEM expression by TNF-α.
    IADR General Session 2011; 03/2011
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    ABSTRACT: Bone morphogenetic proteins (BMPs) possess osteoinductive activities and are useful for clinical treatments, including bone regeneration. We found that transforming growth factor (TGF)-β1 strongly enhances the osteoinductive activity of BMP-2. Collagen sponges containing 5 μg of BMP-2 were implanted into mouse muscle tissues, after which lump-like masses appeared and grew until day 7. Subsequently, calcification occurred in the lump-like masses by day 14. Addition of 50 ng of TGF-β1 to the BMP-2-containing sponges markedly accelerated the growth of the lump-like masses and resulted in a fivefold increase in total bone volume as compared with BMP-2 alone. The number of osteoblasts in ectopic bone tissues at 14 days after implantation induced by BMP-2+TGF-β1 was twofold greater than that with BMP-2 alone, whereas the number of osteoclasts was decreased by half. On the other hand, TGF-β1 accelerated the differentiation of both osteoblasts and osteoclasts in the early stage (2-7 days after implantation) of ectopic bone formation. We also implanted collagen sponges into bone defects surgically created in mouse calvaria. Sponges containing 2.5 μg of BMP-2 and 25 ng of TGF-β1 caused complete filling of the defects with orthotopic bone, whereas those containing 2.5 μg of BMP-2 alone caused only partial filling. These results suggest that TGF-β1 enhances BMP-2-induced ectopic bone formation by accelerating the growth of lump-like masses, and regulates osteoblast and osteoclast generation. Our findings may contribute to the development of a new treatment method for skeletal disorders.
    Tissue Engineering Part A 03/2011; 17(5-6):597-606. DOI:10.1089/ten.TEA.2010.0094 · 4.64 Impact Factor
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    ABSTRACT: Interleukin-1β (IL-1β) induces cell death in chondrocytes in a nitric oxide (NO)- and reactive oxygen species (ROS)-dependent manner. In this study, increased production of lactate was observed in IL-1β-treated mouse chondrocytic ATDC5 cells prior to the onset of their death. IL-1β-induced cell death in ATDC5 cells was suppressed by introducing an siRNA for monocarboxylate transporter-1 (MCT-1), a lactate transporter distributed in plasma and mitochondrial inner membranes. Mct-1 knockdown also prevented IL-1β-induced expression of phagocyte-type NADPH oxidase (NOX-2), an enzyme specialized for production of ROS, whereas it did not have an effect on inducible NO synthase. Suppression of IL-1β-induced cell death by Nox-2 siRNA indicated that NOX-2 is involved in cell death. Phosphorylation and degradation of inhibitor of κBα (IκBα) from 5 to 20 min after the addition of IL-1β was not affected by Mct-1 siRNA. In addition, IκBα was slightly decreased after 12 h of incubation with IL-1β, and the decrease was prominent after 36 h, whereas activation of p65/RelA was observed from 12 to 48 h after exposure to IL-1β. These changes were not seen in Mct-1-silenced cells. Forced expression of IκBα super repressor as well as treatment with the IκB kinase inhibitor BAY 11-7082 suppressed NOX-2 expression. Furthermore, Mct-1 siRNA lowered the level of ROS generated after 15-h exposure to IL-1β, whereas a ROS scavenger, N-acetylcysteine, suppressed both late phase degradation of IκBα and Nox-2 expression. These results suggest that MCT-1 contributes to NOX-2 expression via late phase activation of NF-κB in a ROS-dependent manner in ATDC5 cells exposed to IL-1β.
    Journal of Biological Chemistry 03/2011; 286(17):14744-52. DOI:10.1074/jbc.M111.221259 · 4.60 Impact Factor
  • Rika Yasuhara, Yoichi Miyamoto
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    ABSTRACT: Gingipains are cysteine proteases produced by Porphyromonas gingivalis, one of the major pathogens of periodontitis. They are classified into lysine-specific gingipain (Kgp) and arginine-specific gingipains (Rgps) by the specificity of the proteolytic cutting sites. Gingipains are known to play a major role in the progression of periodontitis by inducing inflammation and tissue destruction in the periodontium, including alveolar bone loss by osteoclasts ; however, the roles of gingipains in osteoclastic bone resorption have not been elucidated yet. Recently, we reported that Kgp but not Rgps and active vitamin D3 or microbial components such as lipopolysaccharide (LPS) synergistically induced osteoclast formation and activation in a setting where both osteoblasts and osteoclast precursor cells co-exist. While LPS has been regarded as one of the major factors for osteoclastogenesis and alveolar bone loss in periodontitis, our findings revealed that not only LPS but also Kgp plays a pivotal role in alveolar bone loss in periodontitis.
    Journal of Oral Biosciences 01/2011; 53(3):197–205. DOI:10.1016/S1349-0079(11)80002-2
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    ABSTRACT: Differentiation of ameloblasts from undifferentiated epithelial cells is controlled by diverse growth factors, as well as interactions between epithelium and mesenchyme. However, there is a considerable lack of knowledge regarding the precise mechanisms that control ameloblast differentiation and enamel biomineralization. We found that the expression level of carbonic anhydrase II (CAII) is strongly up-regulated in parallel with differentiation of enamel epithelium tissues, while the enzyme activity of CA was also increased along with differentiation in ameloblast primary cultures. The expression level of amelogenin, a marker of secretory-stage ameloblasts, was enhanced by ethoxzolamide (EZA), a CA inhibitor, as well as CAII antisense (CAIIAS), whereas the expression of enamel matrix serine proteinase-1 (EMSP-1), a marker for maturation-stage ameloblasts, was suppressed by both. These agents also promoted ameloblast proliferation. In addition, inhibition of ameloblast differentiation by EZA and CAIIAS was confirmed using tooth germ organ cultures. Furthermore, EZA and CAIIAS elevated intracellular pH in ameloblasts, while experimental decreases in intracellular pH abolished the effect of CAIIAS on ameloblasts and triggered the activation of c-Jun N-terminal kinase (JNK). SP600125, a JNK inhibitor, abrogated the response of ameloblasts to an experimental decrease in intracellular pH, while the inhibition of JNK also impaired ameloblast differentiation. These results suggest a novel role for CAII during amelogenesis, that is, controlling the differentiation of ameloblasts. Regulation of intracellular pH, followed by activation of the JNK signaling pathway, may be responsible for the effects of CAII on ameloblasts.
    Journal of Cellular Physiology 12/2010; 225(3):709-19. DOI:10.1002/jcp.22267 · 3.87 Impact Factor

Publication Stats

3k Citations
170.26 Total Impact Points


  • 2006–2014
    • Showa University
      • Department of Biochemistry
      Shinagawa, Tōkyō, Japan
  • 1992–2005
    • Kumamoto University
      • Department of Microbiology
      Kumamoto, Kumamoto Prefecture, Japan
    • JEOL
      Edo, Tōkyō, Japan
  • 2001
    • University of California, Davis
      Davis, California, United States